Immunoeffector compounds

ABSTRACT

This invention provides compounds containing a 2-deoxy-2-amino-β-D-glucopyranose (glucosamine) glycosidically linked to a cyclic aminoalkyl (aglycon) group. The invention further provides methods for inducing an immune response using the compounds of the present invention in the presence or absence of an antigen. In addition, methods for treating disease with the compounds of the present invention with or without an antigen are also provided in this invention.

FIELD OF THE INVENTION

This invention relates generally to immunoeffector compounds, their usein pharmaceutical compositions, and methods for their production andtheir use in prophylactic and/or therapeutic vaccination. Moreparticularly, the present invention relates to an adjuvant systemcomprising 2-deoxy-2-amino-β-D-glucopyranose (glucosamine)glycosidically linked to a cyclic aminoalkyl (aglycon) group.

BACKGROUND OF THE INVENTION

Humoral immunity and cell-mediated immunity are the two major branchesof the mammalian immune response. Humoral immunity involves thegeneration of antibodies to foreign antigens. Antibodies are produced byB-lymphocytes. Cell-mediated immunity involves the activation ofT-lymphocytes which either act upon infected cells bearing foreignantigens or stimulate other cells to act upon infected cells. Bothbranches of the mammalian immune system are important in fightingdisease. Humoral immunity is the major line of defense against bacterialpathogens. In the case of viral disease, the induction of cytotoxic Tlymphocytes (CTLs) appears to be crucial for protective immunity. Thus,an effective vaccine preferably stimulates both branches of the immunesystem to protect against disease.

Vaccines present foreign antigens from disease causing agents to a hostso that the host can mount a protective immune response. Often, vaccineantigens are killed or attenuated forms of the microbes which cause thedisease. The presence of non-essential components and antigens in thesekilled or attenuated vaccines has encouraged considerable efforts torefine vaccine components including developing well-defined syntheticantigens using chemical and recombinant techniques. The refinement andsimplification of microbial vaccines, however, has led to a concomitantloss in potency. Low-molecular weight synthetic antigens, though devoidof potentially harmful contaminants, are often not sufficientlyimmunogenic by themselves. These observations have led investigators toadd immune system stimulators known as adjuvants to vaccine compositionsto potentiate the activity of the vaccine components.

Immune adjuvants are compounds which, when administered to an individualor tested in vitro, increase the immune response to an antigen in asubject to which the antigen is administered, or enhance certainactivities of cells from the immune system. A number of compoundsexhibiting varying degrees of adjuvant activity have been prepared andtested (see, for example, Shimizu et al. 1985, Bulusu et al. 1992, Ikedaet al. 1993, Shimizu et al. 1994, Shimizu et al. 1995, Miyajima et al.1996). However, these and other prior adjuvant systems often displaytoxic properties, are unstable and/or have unacceptably lowimmunostimulatory effects.

Presently, the only adjuvant licensed for human use in the United Statesis alum, a group of aluminum salts (e.g., aluminum hydroxide, aluminumphosphate) in which vaccine antigens are formulated. Particulatecarriers like alum reportedly promote the uptake, processing andpresentation of soluble antigens by macrophages. Alum, however, is notwithout side-effects and is unfortunately limited to humoral (antibody)immunity only.

The discovery and development of effective adjuvant systems is essentialfor improving the efficacy and safety of existing and future vaccines.Thus, there is a continual need for new and improved adjuvant systems,particularly those that drive both effector arms of the immune system,to better facilitate the development of a next generation of syntheticvaccines. The present invention fulfills these and other needs.

SUMMARY OF THE INVENTION

The compounds of the present invention are immunoeffector moleculeswhich enhance humoral and cell-mediated immune responses to vaccineantigens. The compounds comprise a 2-deoxy-2-amino-β-D-glucopyranose(glucosamine) glycosidically linked to an cyclic aminoalkyl (aglycon)group. The compounds are phosphorylated at the 4 or 6-position of theglucosamine ring and acylated with alkanoyloxytetradecanoyl residues onthe aglycon nitrogen and the 2 and 3-positions of the glucosamine ring.The compounds of the subject invention are described generally byformula (I):

and pharmaceutically acceptable salts thereof, wherein X is —O— or —NH—and Y is —O— or —S—; R¹, R², and R³ are each independently a(C₂–C₂₄)acyl group, including saturated, unsaturated and branched acylgroups; R⁴ is —H or —PO₃R⁷R⁸, wherein R⁷ and R⁸ are each independently Hor (C₁–C₄)alkyl; R⁵ is —H, —CH₃ or —PO₃R⁹R¹⁰, wherein R⁹ and R¹⁰ areeach independently selected from —H and (C₁–C₄)alkyl; R⁶ isindependently selected from H, OH, (C₁–C₄)alkoxy, —PO₃R¹¹R¹²,—OPO₃R¹¹R¹², —SO₃R¹¹, —OSO₃R¹¹, —NR¹¹R¹², —SR¹¹, —CN, —NO₂, —CHO,—CO₂R¹¹, and —CONR¹¹R¹², wherein R¹¹ and R¹² are each independentlyselected from H and (C₁–C₄)alkyl; with the proviso that when R⁴ is—PO₃R⁷R⁸, R⁵ is other than —PO₃R⁹R¹⁰, wherein “*¹⁻³” and “**” representchiral centers; wherein the subscripts n, m, p and q are eachindependently an integer from 0 to 6, with the proviso that the sum of pand m is from 0 to 6.

In some embodiments, the compounds of the present invention contain an—O— at X and Y, R⁴ is PO₃R⁷R⁸, R⁵ and R⁶ are H, and the subscripts n, m,p, and q are integers from 0 to 3. In a more preferred embodiment, R⁷and R⁸ are —H. In an even more preferred embodiment, subscript n is 1,subscript m is 2, and subscripts p and q are 0. In yet an even morepreferred embodiment, R₁, R₂, and R₃ are tetradecanoyl residues. In astill more preferred embodiment, *¹⁻³ are in the R configuration, Y isin the equatorial position, and ** is in the S configuration(N—[(R)-3-tetradecanoyloxytetradecanoyl]-(S)-2-pyrrolidinomethyl2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosideand pharmaceutically acceptable salts thereof; RC-553; Formula II).

The present invention also provides compounds which can be used aspharmaceutical compositions containing compounds of the general formulaabove. The pharmaceutical compositions can be combined with a variety ofantigens and in a variety of formulations known to those of skill in theart.

The compounds of the present invention are also useful in methods ofinducing an immune response in a subject. The method entailsadministering a composition containing a therapeutically effectiveamount of a pharmaceutically acceptable carrier and a compound of thepresent invention.

The present invention also encompasses methods of treating a mammalsuffering from or susceptible to a pathogenic infection, cancer or anautoimmune disorder. The method entails administering to the mammal atherapeutically effective amount of a composition containing apharmaceutically acceptable carrier and a compound of the presentinvention.

Still further, the present invention involves a method for treatingdiseases or conditions ameliorated by nitric oxide production in asubject. The method entails contacting the subject with an effectiveamount of a compound of the present invention. In some embodiments, thecompounds of the present invention can be administered 48 hours priorto, up to, and during ischemia.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “acyl” refers to those groups derived from an organic acid byremoval of the hydroxy portion of the acid. Accordingly, acyl is meantto include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl,benzoyl and the like.

A “(C₂–C₂₄)acyl” is an acyl group having from 2 to 24 carbons.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁–C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)ethyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. Alkyl groups which are limited to hydrocarbon groups are termed“homoalkyl.”

A “(C₁–C₄)alkyl” is an alkyl group having from 1 to 4 carbons.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified by—CH₂CH₂CH₂CH₂—, and further includes those groups known as“heteroalkylenes.”

The terms “alkoxy,” “alkylamino” and “alkylthio” refer to those groupshaving an alkyl group attached to the remainder of the molecule throughan oxygen, nitrogen or sulfur atom, respectively.

The term “(C₁–C₄)alkoxy” refers to an alkoxy group having from 1 to 4carbons.

Each of the above terms (e.g., “alkyl,” “acyl”) are meant to includeboth substituted and unsubstituted forms of the indicated radical.Preferred substituents for each type of radical are provided below.

Substituents for the alkyl and acyl radicals can be a variety of groupsselected from: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, —halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂ in a numberranging from zero to (2m′+1), where m′ is the total number of carbonatoms in such radical. R′, R″ and R′″ each independently refer tohydrogen and unsubstituted (C₁–C₈)alkyl. When R′ and R″ are attached tothe same nitrogen atom, they can be combined with the nitrogen atom toform a 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant toinclude 1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and the like.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic,succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge, S. M., et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1–19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Introduction

In an effort to improve the safety of vaccines, manufacturers areavoiding whole cell killed vaccines, and producing recombinant orsubunit vaccines. In the preparation of these safer vaccines extraneousbacterial or viral components are eliminated, while the minimalstructures or epitopes deemed necessary for protective immunity remain.The safety of these vaccines is improved due to the elimination ofextraneous bacterial or viral components which often times prove to betoxic and pyrogenic. However, the same components that result intoxicity provide nonspecific immunostimulation that make whole cellvaccines so effective. Without the additional immunostimulation theminimal structures and epitopes comprising recombinant and subunitvaccines are often poorly immunogenic.

A disaccharide molecule derived from the LPS of Salmonella minnesotaR595, MPL® immunostimulant (Corixa Corp.), has immunostimulantproperties. MPL® immunostimulant, Monophosphoryl lipid A, is astructural derivative of lipid A (or LPS) and has an improvedtherapeutic index relative to lipid A (see U.S. Pat. No. 4,987,237 forthe structure of Monophosphoryl lipid A; U.S. Pat. Nos. 4,436,727 and4,436,728 for description of preparation of Monophosphoryl lipid A).Other useful immunostimulants include 3-de-O-acylated monophosphoryllipid A (3D-MPL), which is described in U.S. Pat. No. 4,912,094. Thecompound can be safely administered to humans as doses up to at least 20μg/kg, although increases in temperature, flu-like symptoms, increasingheart rate and modest decreases in blood pressure can occur in somepatients at dose levels of ≦10 μg/kg. Cell culture and animalevaluations confirm MPL® immunostimulant still retains some of theimmunostimulatory activity of the parent LPS in that pyrogenicity andthe ability to induce pro-inflammatory cytokines such as TNF and IL-8remain, albeit at higher dose levels. Thus, the need for effectivevaccine adjuvants is well recognized. Ideally, these adjuvants willboost the protective immune response without inducing unwanted toxicityand pyrogenicity.

In an effort to obtain an immunostimulant that has low pyrogenicity,synthetic molecules have been prepared which share structuralsimilarities with the MPL® immunostimulant (see U.S. patent applicationSer. No. 09/810,915, Filed on Mar. 16, 2001, and the present invention).These novel molecules which are collectively called aminoalkylglucosaminide phosphates (AGPs), consist of an acylated glucose moietylinked to an acylated aminoalkyl group (Johnson et al. (1999) Bioorg.Med. Chem. Lett. 9: 2273–2278; PCT/WO98/50399 and references therein).Each molecule possesses 6 fatty acid tails which is thought to be theoptimal number for peak adjuvant activity. The substitution of differentchemical moieties within the aminoalkyl structures was designed into theAGPs in anticipation of optimizing stability and solubility properties.Thus the AGPs can be broadly separated into several families based onthe structure of their aminoalkyl groups. After initial biologicalevaluation, it became apparent that the aminoalkyl motifs coulddramatically affect the pyrogenic properties of the AGPs (see U.S.patent application Ser. Nos. 09/810915 (filed on Mar. 16, 2001),09/439,839, 09/074,720 and U.S. Pat. No. 6,113,918 (which issued fromU.S. Ser. No. 08/853,826). As part of the initial screening process ofthe synthetic adjuvant compounds, rabbit pyrogenicity data wasdetermined. It was noted that several of the compounds did not elicit afever response when administered i.v. at doses of 10 μg/kg. In general,these same compounds failed to induce detectable levels of inflammatorycytokines TNF-α or IL-1β in an ex vivo cytokine induction assay on humanperipheral blood mononuclear cells. Here we report on studies of theadjuvant properties of a class of AGPs which induce minimal activity inboth the rabbit pyrogen test and the ex vivo cytokine assay.

Compounds and Compositions

The present invention provides compounds described generally by formula(I):

and pharmaceutically acceptable salts thereof, wherein X is —O— or —NH—and Y is —O— or —S—; R¹, R², and R³ are each independently a(C₂–C₂₄)acyl group, including saturated, unsaturated and branched acylgroups; R⁴ is —H or —PO₃R⁷R⁸, wherein R⁷ and R⁸ are each independently Hor (C₁–C₄)alkyl; R⁵ is —H, —CH₃ or —PO₃R⁹R¹⁰, wherein R⁹ and R¹⁰ areeach independently selected from —H and (C₁–C₄)alkyl; R⁶ isindependently selected from H, OH, (C₁–C₄)alkoxy, —PO₃R¹¹R¹²,—OPO₃R¹¹R¹², —SO₃R¹¹, —OSO₃R¹¹, —NR¹¹R¹², —SR¹¹, —CN, —NO₂, —CHO,—CO₂R¹¹, and —CONR¹¹R¹², wherein R¹¹ and R¹² are each independentlyselected from H and (C₁–C₄)alkyl; with the proviso that when R⁴ is—PO₃R⁷R⁸, R⁵ is other than —PO₃R⁹R¹⁰, wherein “*¹⁻³” and “**” representchiral centers; wherein the subscripts n, m, p and q are eachindependently an integer from 0 to 6, with the proviso that the sum of pand m is from 0 to 6.

Although the hexopyranoside in Formula I is shown in the glucoconfiguration, other glycosides are within the scope of the invention.For example glycopyranosides, including other hexopyranosides (e.g.,allo, altro, manno, gulo, ido, galacto, talo), are within the scope ofthe invention.

In the general formula above, the configuration of the 3′-stereogeniccenters to which the normal fatty acyl residues are attached, denoted“*¹”, “*²” and “*³”, is R or S, but preferably R. The absolutestereochemistry of the carbon atoms of the cyclic aglycon unit to whichR⁶ and the glucosamine unit are attached, directly or indirectly(denoted “**”) can be R or S. In the general formula above, Y can be inthe equatorial or axial position, but is preferably equatorial. Allstereoisomers, enantiomers, diastereomers and mixtures thereof areconsidered to be within the scope of the present invention.

In preferred embodiments, of the present invention, X and Y are —O—, R⁴is phosphono, R⁵ and R⁶ are H, and the subscripts n, m, p, and q areintegers of from 0 to 3, and more preferably 0 to 2. Most preferably theinteger n is 1, the integer m is 2, and integers p and q are 0. In thispreferred embodiment, the compounds of this invention are2-pyrrolidinomethyl β-D-glucosaminide 4-phosphates having the generalformula (III):

In the most preferred embodiment of the present invention, R₁, R₂, andR₃ of formula (III) are tetradecanoyl residues and the configuration ofthe 3′-stereogenic centers (“*¹⁻³”) to which they are attached is R, Yis in the equatorial position, and the absolute stereochemistry of thepyrrolidine stereogenic center (“**”) is S. Specifically, the compoundof the most preferred embodiment isN—[(R)-3-tetradecanoyloxytetradecanoyl]-(S)-2-pyrrolidinomethyl2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosideand pharmaceutically acceptable salts thereof. This most preferredembodiment is also known as RC-553 and is depicted in formula II:

Preparation of Compounds

The compounds of the present invention can be prepared using methodsoutlined in Johnson et al., Bioorg. Med. Chem. Lett. 9:2273–2278 (1999)and PCT/WO98/50399 and references therein. In general, the syntheticmethods described in the above-noted references are broadly applicableto the preparation of compounds having different acyl groups andsubstitutions. One of skill in the art will appreciate that theconvergent methods described therein can be modified to use alternateacylating agents, or can be initiated with commercially availablematerials having appropriate acyl groups attached.

Evaluation of Compounds

The compounds provided herein can be evaluated in a variety of assayformats to select a compound having a suitable pharmacophoric profile.For example, U.S. Pat. No. 6,013,640 describes animal models suitablefor evaluating cardioprotective effects of compounds described herein.The examples below also provide assays for evaluating pyrogenicity ofthe subject compounds, and further assays for evaluating theproinflammatory effects of the compounds.

The present invention further provides pharmaceutical compositionscomprising the compounds provided herein in admixture with one or morepharmaceutically acceptable carriers. Suitable carriers will depend onthe condition being treated along with the route of administration.Accordingly, a discussion of the carriers is provided below inconjunction with the methods of use.

Pharmaceutical Compositions and Their Uses

In one embodiment, the present invention provides pharmaceuticalcompositions containing a compound of the present invention and apharmaceutically acceptable carrier. The compound is present in atherapeutically effective amount, which the amount of compound requiredto achieve the desired effect in terms of treating a disease, condition,or achieving a biological occurrence. The pharmaceutical compositionscan act as an adjuvant when co-administered with an antigen.

Thus, the adjuvant systems of the invention are particularlyadvantageous in making and using vaccine and other immunostimulantcompositions to treat or prevent diseases, such inducing active immunitytowards antigens in mammals, preferably in humans. Vaccine preparationis a well developed art and general guidance in the preparation andformulation of vaccines is readily available from any of a variety ofsources. One such example is New Trends and Developments in Vaccines,edited by Voller et al., University Park Press, Baltimore, Md., U.S.A.1978.

In one illustrative embodiment, the antigen in a vaccine composition ofthe invention is a peptide, polypeptide, or immunogenic portion thereof.An “immunogenic portion,” as used herein is a portion of a protein thatis recognized (i.e., specifically bound) by a B-cell and/or T-cellsurface antigen receptor. Such immunogenic portions generally compriseat least 5 amino acid residues, more preferably at least 10, and stillmore preferably at least 20 amino acid residues of an antigenic proteinor a variant thereof.

Immunogenic portions of antigen polypeptides may generally be identifiedusing well known techniques, such as those summarized in Paul,Fundamental Immunology, 3rd ed., 243–247 (Raven Press, 1993) andreferences cited therein. Such techniques include screening polypeptidesfor the ability to react with antigen-specific antibodies, antiseraand/or T-cell lines or clones. As used herein, antisera and antibodiesare “antigen-specific” if they specifically bind to an antigen (i.e.,they react with the protein in an ELISA or other immunoassay, and do notreact detectably with unrelated proteins). Such antisera and antibodiesmay be prepared as described herein, and using well known techniques. Animmunogenic portion of a protein is a portion that reacts with suchantisera and/or T-cells at a level that is not substantially less thanthe reactivity of the full length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Such immunogenic portions may react withinsuch assays at a level that is similar to or greater than the reactivityof the full length polypeptide. Such screens may generally be performedusing methods well known to those of ordinary skill in the art, such asthose described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may beimmobilized on a solid support and contacted with patient sera to allowbinding of antibodies within the sera to the immobilized polypeptide.Unbound sera may then be removed and bound antibodies detected using,for example, ¹²⁵I-labeled Protein A.

Peptide and polypeptide antigens are prepared using any of a variety ofwell-known techniques. Recombinant polypeptides encoded by DNA sequencesmay be readily prepared from isolated DNA sequences using any of avariety of expression vectors known to those of ordinary skill in theart. Expression may be achieved in any appropriate host cell that hasbeen transformed or transfected with an expression vector containing aDNA molecule that encodes a recombinant polypeptide. Suitable host cellsinclude prokaryotes, yeast, and higher eukaryotic cells, such asmammalian cells and plant cells. Preferably, the host cells employed areE. coli, yeast or a mammalian cell line such as COS or CHO.

Portions and other variants of a protein antigen having less than about100 amino acids, and generally less than about 50 amino acids, may alsobe generated by synthetic means, using techniques well known to those ofordinary skill in the art. For example, such polypeptides may besynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain. SeeMerrifield, J. Am. Chem. Soc. 85:2149–2146, 1963. Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystems Division (Foster City,Calif.), and may be operated according to the manufacturer'sinstructions.

Within certain specific embodiments, a polypeptide antigen used in thevaccine compositions of the invention may be a fusion protein thatcomprises two or more distinct polypeptides. A fusion partner may, forexample, assist in providing T helper epitopes (an immunological fusionpartner), preferably T helper epitopes recognized by humans, or mayassist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of the protein.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein, allowing the production of increasedlevels, relative to a non-fused protein, in an expression system.Briefly, DNA sequences encoding the polypeptide components may beassembled separately, and ligated into an appropriate expression vector.The 3′ end of the DNA sequence encoding one polypeptide component isligated, with or without a peptide linker, to the 5′ end of a DNAsequence encoding the second polypeptide component so that the readingframes of the sequences are in phase. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39–46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258–8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

Within preferred embodiments, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100–110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemagglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265–292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795–798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188–305.

In another embodiment of the invention, the adjuvant system describedherein is used in the preparation of DNA-based vaccine compositions.Illustrative vaccines of this type contain DNA encoding one or morepolypeptide antigens, such that the antigen is generated in situ. TheDNA may be present within any of a variety of delivery systems known tothose of ordinary skill in the art, including nucleic acid expressionsystems, bacteria and viral expression systems. Numerous gene deliverytechniques are well known in the art, such as those described byRolland, Crit. Rev. Therap. Drug Carrier Systems 15:143–198, 1998, andreferences cited therein. Appropriate nucleic acid expression systemscontain the necessary DNA sequences for expression in the patient (suchas a suitable promoter and terminating signal). Bacterial deliverysystems involve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope. In onepreferred embodiment, the DNA is introduced using a viral expressionsystem (e.g., vaccinia or other pox virus, retrovirus, or adenovirus),which typically involves the use of a non-pathogenic (defective),replication competent virus. Illustrative systems are disclosed, forexample, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317–321,1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86–103, 1989; Flexner etal., Vaccine 8:17–21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; Berkner, Biotechniques 6:616–627, 1988;Rosenfeld et al., Science 252:431–434, 1991; Kolls et al., Proc. Natl.Acad. Sci. USA 91:215–219, 1994; Kass-Eisler et al., Proc. Natl. Acad.Sci. USA 90:11498–11502, 1993; Guzman et al., Circulation 88:2838–2848,1993; and Guzman et al., Cir. Res. 73:1202–1207, 1993. Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art.

Alternatively, the DNA may be “naked,” as described, for example, inUlmer et al., Science 259:1745–1749, 1993 and reviewed by Cohen, Science259:1691–1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads that are efficiently transported intothe cells. It will be apparent that a vaccine may comprise both apolynucleotide and a polypeptide component if desired.

Moreover, it will be apparent that a vaccine may containpharmaceutically acceptable salts of the desired polynucleotide,polypeptide and/or carbohydrate antigens. For example, such salts may beprepared from pharmaceutically acceptable non-toxic bases, includingorganic bases (e.g., salts of primary, secondary and tertiary amines andbasic amino acids) and inorganic bases (e.g., sodium, potassium,lithium, ammonium, calcium and magnesium salts).

The adjuvant system of the present invention exhibits strong adjuvanteffects when administered over a wide range of dosages and a wide rangeof ratios.

The amount of antigen in each vaccine dose is generally selected as anamount which induces an immunoprotective response without significantadverse side effects in typical vaccines. Such amount will varydepending upon which specific immunogen is employed and how it ispresented. Generally, it is expected that each dose will comprise about1–1000 μg of protein, most typically about 2–100 μg, preferably about5–50 μg. Of course, the dosage administered may be dependent upon theage, weight, kind of concurrent treatment, if any, and nature of theantigen administered.

The immunogenic activity of a given amount of a vaccine composition ofthe present invention can be readily determined, for example bymonitoring the increase in titer of antibody against the antigen used inthe vaccine composition (Dalsgaard, K. Acta Veterinia Scandinavica69:1–40 (1978)). Another common method involves injecting CD-1 miceintradermally with various amounts of a vaccine composition, laterharvesting sera from the mice and testing for anti-immunogen antibody,e.g., by ELISA. These and other similar approaches will be apparent tothe skilled artisan.

The antigen can be derived and/or isolated from essentially any desiredsource depending on the infectious disease, autoimmune disease,condition, cancer, pathogen, or a disease that is to be treated with agiven vaccine composition. By way of illustration, the antigens can bederived from viral sources, such as influenza virus, feline leukemiavirus, feline immunodeficiency virus, Human HIV-1, HIV-2, Herpes Simplexvirus type 2, Human cytomegalovirus, Hepatitis A, B, C or E, RespiratorySyncytial virus, human papilloma virus rabies, measles, or hoof andmouth disease viruses. Illustrative antigens can also be derived frombacterial sources, such as anthrax, diphtheria, Lyme disease, malaria,tuberculosis, Leishmaniasis, T. cruzi, Ehrlichia, Candida etc., or fromprotozoans such as Babeosis bovis or Plasmodium. The antigen(s) willtypically be comprised of natural or synthetic amino acids, e.g., in theform of peptides, polypeptides, or proteins, can be comprised ofpolysaccharides, or can be mixtures thereof. Illustrative antigens canbe isolated from natural sources, synthesized by means of solid phasesynthesis, or can be obtained by way of recombinant DNA techniques.

In another embodiment, tumor antigens are used in the vaccinecompositions of the present invention for the prophylaxis and/or therapyof cancer. Cancer cells often have distinctive antigens on theirsurfaces, such as truncated epidermal growth factor, folate bindingprotein, epithelial mucins, melanoferrin, carcinoembryonic antigen,prostate-specific membrane antigen, HER2-neu, which are candidates foruse in therapeutic cancer vaccines. Because tumor antigens are normal orrelated to normal components of the body, the immune system often failsto mount an effective immune response against those antigens to destroythe tumor cells. To achieve such a response, the adjuvant systemsdescribed herein can be utilized. As a result, exogenous proteins canenter the pathway for processing endogenous antigens, leading to theproduction of cytolytic or cytotoxic T cells (CTL). This adjuvant effectfacilitates the production of antigen specific CTLs which seek anddestroy those tumor cells carrying on their surface the tumor antigen(s)used for immunization. Illustrative cancer types for which this approachcan be used include prostate, colon, breast, ovarian, pancreatic, brain,head and neck, melanoma, leukemia, lymphoma, etc.

In another embodiment of the invention, the adjuvant system of thepresent invention can be administered alone, i.e., without aco-administered antigen, to potentiate the immune system for treatmentof chronic infectious diseases, especially in immune compromisedpatients. Illustrative examples of infectious diseases for which thisapproach may be employed for therapeutic or prophylactic treatment canbe found in U.S. Pat. No. 5,508,310. Potentiation of the immune systemin this way can also be useful as a preventative measure to limit therisks of nosocomial and/or post-surgery infections.

In another embodiment, the antigen present in the vaccine compositionsis not a foreign antigen, rather it is a self antigen, e.g., the vaccinecomposition is directed toward an autoimmune disease such as type 1diabetes, conventional organ-specific autoimmune diseases, neurologicaldiseases, rheumatic diseases, psoriasis, connective tissue diseases,autoimmune cytopenias, and other autoimmune diseases. Such conventionalorgan specific autoimmunity may include thyroiditis(Graves+Hashimoto's), gastritis, adrenalitis (Addison's), ovaritis,primary biliary cirrhosis, myasthenia gravis, gonadal failure,hypoparathyroidism, alopecia, malabsorption syndrome, pernicious anemia,hepatitis, anti-receptor antibody diseases and vitiligo. Suchneurological diseases may include schizophrenia, Alzheimer's disease,depression, hypopituitarism, diabetes insipidus, sicca syndrome andmultiple sclerosis. Such rheumatic diseases/connective tissue diseasesmay include rheumatoid arthritis, systemic lupus erythematous (SLE) orLupus, scleroderma, polymyositis, inflammatory bowel disease,dermatomyositis, ulcerative colitis, Crohn's disease, vasculitis,psoriatic arthritis, exfoliative psoriatic dermatitis, pemphigusvulgaris, Sjogren's syndrome. Other autoimmune related diseases mayinclude autoimmune uvoretinitis, glomerulonephritis, post myocardialinfarction cardiotomy syndrome, pulmonary hemosiderosis, amyloidosis,sarcoidosis, aphthous stomatitis, and other immune related diseases, aspresented herein and known in the related arts.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the vaccine compositions of this invention, the typeof carrier will typically vary depending on the desired mode ofadministration. Compositions of the present invention may be formulatedfor any appropriate manner of administration, including for example,topical, oral, nasal, intravenous, intracranial, intraperitoneal,intradermal, subcutaneous or intramuscular administration. Forparenteral administration, such as subcutaneous injection, the carrierwill often comprise water, saline, alcohol, a fat, a wax or a buffer.For oral administration, the above carriers are often used, or a solidcarrier such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate, can also be employed. Biodegradable microspheres (e.g.,polylactate polyglycolate) may also be employed as carriers for thecompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252, thedisclosures of which are incorporated herein by reference in theirentireties. Modified hepatitis B core protein carrier systems are alsosuitable, such as those described in WO/99 40934, and references citedtherein, all incorporated herein by reference. One can also employ acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, the disclosure of which isincorporated herein by reference in its entirety, which are capable ofinducing a class I-restricted cytotoxic T lymphocyte responses in ahost.

In one illustrative embodiment, the vaccine formulations areadministered to the mucosae, in particular to the oral cavity, andpreferably to a sublingual site, for eliciting an immune response. Oralcavity administration may be preferred in many instances overtraditional parenteral delivery due to the ease and convenience offeredby noninvasive administration techniques. Moreover, this approachfurther provides a means for eliciting mucosal immunity, which can oftenbe difficult to achieve with traditional parenteral delivery, and whichcan provide protection from airborne pathogens and/or allergens. Anadditional advantage of oral cavity administration is that patientcompliance may be improved with sublingual vaccine delivery, especiallyfor pediatric applications, or for applications traditionally requiringnumerous injections over a prolonged period of time, such as withallergy desensitization therapies.

The vaccine compositions can also comprise buffers (e.g. neutralbuffered saline, phosphate buffered saline or phosphate buffers w/osaline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans),mannitol, proteins, polypeptides or amnino acids such as glycine,antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate. The compositions can also beencapsulated within liposomes using well known technology.

Therefore, in one embodiment, the vaccine compositions are aqueousformulations comprising an effective amount of one or more surfactants.For example, the composition can be in the form of a micellar dispersioncomprising at least one suitable surfactant, e.g., a phospholipidsurfactant. Illustrative examples of phospholipids include diacylphosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol(DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoylphosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such asdimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine(DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidicacids, such as dimyristoyl phosphatidic acid (DPMA), dipalmitoylphosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); anddiacyl phosphatidyl ethanolamines such as diimyristoyl phosphatidylethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine (DPPE) anddistearoyl phosphatidyl ethanolamine (DSPE).

Typically, a surfactant:adjuvant molar ratio in an aqueous formulationwill be from about 10:1 to about 1:10, more typically from about 5:1 toabout 1:5, however any effective amount of surfactant may be used in anaqueous formulation to best suit the specific objectives of interest.

In another embodiment, the composition is an emulsion, such as awater-in-oil emulsion or an oil-in water emulsion. Such emulsions aregenerally well known to those skilled in this art.

The adjuvant system of the present invention can be employed as the soleadjuvant system, or alternatively, can be administered together withother adjuvants or immunoeffectors. By way of illustration, suchadjuvants can include oil-based adjuvants (for example, Freund'sComplete and Incomplete), liposomes, mineral salts (for example,AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica, alum, Al(OH)₃, Ca₃(PO₄)₂,kaolin, and carbon), polynucleotides (for example, poly IC and poly AUacids), polymers (for example, non-ionic block polymers,polyphosphazenes, cyanoacrylates, polymerase-(DL-lactide-co-glycoside),among others, and certain natural substances (for example, lipid A andits derivatives, wax D from Mycobacterium tuberculosis, as well assubstances found in Corynebacterium parvum, Bordetella pertussis, andmembers of the genus Brucella), bovine serum albumin, diphtheria toxoid,tetanus toxoid, edestin, keyhole-limpet hemocyanin, Pseudomonal Toxin A,choleragenoid, cholera toxin, pertussis toxin, viral proteins, andeukaryotic proteins such as interferons, interleukins, or tumor necrosisfactor. Such proteins may be obtained from natural or recombinantsources according to methods well known to those skilled in the art.When obtained from recombinant sources, the adjuvant may comprise aprotein fragment comprising at least the immunostimulatory portion ofthe molecule. Other known immunostimulatory macromolecules which can beused in the practice of the invention include, but are not limited to,polysaccharides, tRNA, non-metabolizable synthetic polymers such aspolyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixedpolycondensates (with relatively high molecular weight) of4′,4-diaminodiphenylmethane-3,3′-dicarboxylic acid and4-nitro-2-aminobenzoic acid (See Sela, M., Science 166:1365–1374 (1969))or glycolipids, lipids or carbohydrates.

In one embodiment, the adjuvant system is preferably designed to inducean immune response predominantly of the Th1 type. High levels ofTh1-type cytokines (e.g., IFN-γ, TNFα, IL-2 and IL-12) tend to favor theinduction of cell mediated immune responses to an administered antigen.In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6and IL-10) tend to favor the induction of humoral immune responses.Following application of a vaccine as provided herein, a patient willsupport an immune response that includes Th1- and Th2-type responses.Within a preferred embodiment, in which a response is predominantlyTh1-type, the level of Th1-type cytokines will increase to a greaterextent than the level of Th2-type cytokines. The levels of thesecytokines may be readily assessed using standard assays. For a review ofthe families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol.7:145–173, 1989.

For example, additional adjuvants for use in eliciting a predominantlyTh1-type response include, for example, a combination of monophosphoryllipid A, such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL),together with an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Other illustrativeadjuvants that can be included in the vaccine compositions includeMontanide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States),ISCOMS (CSL), MF-59 (Chiron), Detox™ adjuvant (Corixa, Hamilton, Mont.).

The compositions described herein may be administered as part of asustained release formulation (ie., a formulation such as a capsule,sponge or gel (composed of polysaccharides, for example) that effects aslow release of compound following administration). Such formulationsmay generally be prepared using well known technology (see, e.g.,Coombes et al., Vaccine 14:1429–1438, 1996) and administered by, forexample, oral, rectal or subcutaneous implantation, or by implantationat the desired target site. Sustained-release formulations may contain apolypeptide, polynucleotide or antibody dispersed in a carrier matrixand/or contained within a reservoir surrounded by a rate controllingmembrane. Carriers for use within such formulations are biocompatible,and may also be biodegradable; preferably the formulation provides arelatively constant level of active component release. Such carriersinclude microparticles of poly(lactide-co-glycolide), polyacrylate,latex, starch, cellulose, dextran and the like. Other delayed-releasecarriers include supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation will vary depending upon the site of implantation,the rate and expected duration of release and the nature of thecondition to be treated or prevented.

Any of a variety of known delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets cells. Delivery vehiclesinclude antigen presenting cells (APCs), such as dendritic cells,macrophages, B cells, monocytes and other cells that may be engineeredto be efficient APCs. Such cells may, but need not, be geneticallymodified to increase the capacity for presenting the antigen, to improveactivation and/or maintenance of the T cell response, to haveanti-target effects per se and/or to be immunologically compatible withthe receiver (i.e., matched HLA haplotype). APCs may generally beisolated from any of a variety of biological fluids an organs, includingtumor and peritumoral tissues, and may be autologous, allogeneic,syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245–251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507–529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594–600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g. CD40, CD80, CD86and 4-1 BB).

APCs may generally be transfected with a polynucleotide encoding anantigen polypeptide (or portion or other variant thereof) such that theantigen polypeptide, or an immunogenic portion thereof, is expressed onthe cell surface. Such transfection may take place ex vivo, and acomposition or vaccine comprising such transfected cells, and theadjuvants described herein, may then be used for therapeutic purposes.Alternatively, a gene delivery vehicle that targets a dendritic or otherantigen presenting cell may be administered to a patient, resulting intransfection that occurs in vivo. In vivo and ex vivo transfection ofdendritic cells, for example, may generally be performed using anymethods known in the art, such as those described in WO 97/24447, or thegene gun approach described by Mahvi et al., Immunology and cell Biology75:456460, 1997. Antigen loading of dendritic cells may be achieved byincubating dendritic cells or progenitor cells with the antigenpolypeptide, DNA (naked or within a plasmid vector) or RNA; or withantigen-expressing recombinant bacterium or viruses (e.g. vaccinia,fowlpox, adenovirus or lentivirus vectors). Prior to loading, thepolypeptide may be covalently conjugated to an immunological partnerthat provides T cell help (e.g., a carrier molecule). Alternatively, adendritic cell may be pulsed with a non-conjugated immunologicalpartner, separately or in the presence of the polypeptide.

Treatment of Nitric Oxide Related Disorders

In one aspect, the present invention provides methods for treatingdiseases or conditions mediated by nitric oxide, particularly ischemiaand reperfusion injury. The methods comprise administering to a subjectin need of such treatment an effective amount of a compound of thepresent invention. It is generally agreed that inducers of iNOS genetranscription and protein synthesis are proinflammatory and consequentlysomewhat “toxic” or poorly tolerated in animals and humans. Endotoxin(LPS) and proinflammatory cytokines such as IL-1, TNF-α and IFN-γ areknown inducers of iNOS. All are inherently toxic and capable of inducinga systemic inflammatory response, adult respiratory distress syndrome,multiple organ failure and cardiovascular collapse when administered toanimals.

Investigation of the cardioprotective activity of MPL® immunostimulantdemonstrated that induction of nitric oxide synthases (iNOS) isimportant in the delayed cardioprotective effect of the compound.Additionally, nitric oxide (NO) signaling, presumably throughconstitutive pools of NOS, is important in the acute cardioprotectiveeffect of the compound. In view of the residual endotoxic-like activityof MPL® immunostimulant, it is not surprising that the compound could becapable of inducing nitric oxide signaling. Still further, nitric oxidesignaling has been suggested as a potential pathway by which ischemicpreconditioning elicits cardioprotection. This observation incombination with the fact that nitric oxide donors are cardioprotectiveprovides further support for the NOS/NO pathway as the route for MPL®immunostimulant cardioprotection.

The compounds of the present invention, including RC-553, are useful inmethods for treating diseases or conditions modulated or ameliorated bynitric oxide, particularly ischemia and reperfusion injury (see, U.S.patent application Ser. No. 09/808669, filed Mar. 14, 2001, for adescription of the cardioprotective properties of aminoalkylglucosaminide phosphates and methods for assaying cardioprotectiveproperties).

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Preparation of RC-553

Preparation ofN—[(R)-3-Tetradecanoyloxytetradecanoyl]-(S)-2-pyrrolidinomethyl2-Deoxy4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (formula (I), R₁═R₂═R₃═C¹³H₂₇CO, X═Y═O, n=1, m=2,p=q=0, R₅═R₆═H, R₄═PO₃H₂; formula (II) R₁═R₂═R₃═C₁₃H₂₇CO); RC-553.

(1) To a solution of2-deoxy-4-O-diphenylphosphono-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosylbromide (1.05 g, 0.81 mmol) in dry 1,2-dichloroethane (10 mL) were added4 Å molecular sieves (0.5 g), anhydrous CaSO₄ (2.2 g, 16 mmol), andN—[(R)-3-tetradecanoyloxytetradecanoyl]-(S)-2-pyrrolidinomethanol (0.40g, 0.75 mmol). The resulting mixture was stirred for 1 h at roomtemperature, treated with Hg(CN)₂ (1.02 g, 4.05 mmol), and heated toreflux for 16 h in the dark. The cooled reaction mixture was dilutedwith CH₂Cl₂ and filtered. The filtrate was washed with 1 N aq KI, dried(Na₂SO₄), and concentrated. Flash chromatography on silica gel (gradientelution, 15→20% EtOAc/hexanes) afforded 0.605 g (43%) ofN—-[(R)-3-tetradecanoyloxytetradecanoyl]-(S)-2-pyrrolidinomethyl2-deoxy-4-O-diphenylphosphono-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid.

(2) A solution of the compound prepared in (1) above (0.50 g, 0.29 mmol)in AcOH (10 mL) at 60° C. was treated with zinc dust (0.98 g, 15 mmol)in three equal portions over a 1-h period; The cooled reaction mixturewas sonicated, filtered through a pad of Celite, and concentrated. Theresulting residue was partitioned between CH₂Cl₂ and saturated aqNaHCO₃, and the layers were separated. The organic layer was dried(Na₂SO₄) and concentrated. A solution of the crude amino alcoholobtained and (R)-3-tetradecanoyloxytetradecanoic acid (0.155 g, 0.34mmol) in CH₂Cl₂ (3.5 mL) was stirred with powdered 4 Å molecular sieves(0.25 g) for 0.5 h and then treated with2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (0.11 g, 0.44 mmol). Theresulting mixture was stirred at room temperature for 8 h, filteredthrough Celite, and concentrated. Flash chromatography on silica gelwith 50% EtOAc/hexanes gave 0.355 g (68%) ofN—[(R)-3-tetradecanoyloxytetradecanoyl]-(S)-2-pyrrolidinomethyl2-deoxy-4-O-diphenylphosphono-2—[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosideas a colorless syrup.

(3) A solution of the compound prepared in (2) above (0.300 g, 0.166mmol) in a mixture of AcOH (1 mL) and tetrahydrofuran (9 mL) washydrogenated in the presence of PtO₂ (0.15 g) at room temperature and 70psig for 18 h. The reaction mixture was diluted with 2:1 CHCl₃-MeOH (50mL) and sonicated briefly. The catalyst was collected and washed with2:1 CHCl₃-MeOH and the combined filtrate and washings were concentrated.Flash chromatography on silica gel with CHCl₃—MeOH—H₂O-Et₃N(90:10:0.5:0.5) gave partially purified product which was dissolved inice-cold 2:1 CHCl₃—MeOH (30 mL) and washed with ice-cold 0.1 N aq HCl(12 mL). The organic phase was filtered and lyophilized from 2% aq Et₃N(5 mL, pyrogen-free) to give 0.228 g (79%) ofN—[(R)-3-tetradecanoyloxytetradecanoyl]-(S)-2-pyrrolidinomethyl2-deoxy-4-O-phosphono-2—[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a colorless powder: mp 67–70° C.; IR (film)3306, 2955, 2923, 2853, 1736, 1732, 1644, 1548, 1466, 1378, 1245, 1177,1110, 1053, 844 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (m, 18H), 1.0–1.2.05(mH), 2.20–2.70 (m, 12H), 3.06 (q, 6H, J=7.2 Hz), 3.3–325 (mH), 4.52 (d,1H, J=8 Hz), 5.05–5.28 (m, 4H), 7.44 (d, 1H, J=9 Hz); ¹³C NMR (CDCl₃) δ173.3, 173.0, 170.3, 169.6, 168.6, 101.8, 100.4, 75.8, 72.5, 72.4, 70.9,70.8, 70.3, 70.2, 69.9, 69.3, 67.9, 66.6, 56.5, 56.3, 54.5, 47.4, 45.8,44.6, 41.4, 41.0, 39.7, 39.2, 39.0, 34.5, 34.3, 34.1, 32.0, 29.7, 29.4,28.1, 27.3, 25.7, 25.3, 25.2, 25.1, 24.0, 22.7, 21.6, 14.1, 8.6.

Anal. Calcd. for C₁₀₁H₁₉₄N₃O₁₇P.H₂O: C, 68.47; H, 11.15; N, 2.37; P,1.75. Found: C, 68.79; H, 11.00; N, 2.24; P, 1.97.

Examples 2–6

The primary objective of Examples 2–6 was to determine if RC-553 couldpromote minimal pyrogenicity and mediate adjuvant activity whenformulated with vaccine antigens.

Example 2 Adjuvant Activity Towards HBsAg (Hepatitis B Surface Antigen)

Groups of BALB/c mice (Jackson Laboratories Bar Harbor, Me.) 6–8 weeksold were injected s.c. with 2 μg HBsAg (Laboratorio Pablo Cassara) ±20μg adjuvant (MPL® immunostimulant or RC-553) on day 0 and day 21.Vaccines were prepared by mixing the adjuvant-containing TEoA(triethanolamine) formulations with recombinant HBsAg. Titers to HBsAgwere determined by ELISA from pooled sera (5 mice/group) collected 21days post-secondary vaccination (Table 1). The nonimmune controls werenot vaccinated.

Serum titers from mice receiving RC-553 had anti-HBsAg responsessignificantly higher than control sera receiving antigen alone (Table1). Especially noticeable was the increase in the titers for the IgG2aand IgG2b isotypes. These titers were equivalent to those expressed bycontrol groups receiving MPL® immunostimulant.

TABLE 1 Comparison of Low Pyrogen Adjuvants with HBsAg Pyrogen- SerumTiters Groups icity^(a) IgG IgG1 IgG2a IgG2b Nonimmune — <100 <100 <100<100 TEoA Vehicle N.T. 51,200 102,400 25,600 1600 MPL ®-TEoA 2–3 409,600204,800 204,800 51,200 RC-553-TEoA 0.3 409,600 204,800 409,600 51,200^(a)The pyrogenicity data represents the total rise in ° C. of 3 rabbitsfollowing i.v. administration of a 10 μg/Kg dose. In the pyrogen assaythe compounds were solubilized in 10% EtOH/WFI(USP Water for Injection)at 100 μg/ml and then diluted with 5% dextrose in water. N.T. means thecompound was not tested.

Example 3 Adjuvant Activity Towards Hemagglutinin Protein in FluZoneInfluenza Vaccine

Groups of BALB/c mice (Jackson Laboratories Bar Harbor, Me.) 6–8 weeksold were injected subcutaneous with 0.2 μg hemagglutinin protein inFluZone influenza vaccine (Connaught Laboratories, Swiftwater, Pa.) ±20μg adjuvant (MPL® immunostimulant or RC-553) on day 0 and day 14. Titersto FluZone were determined by FluZone ELISA from pooled sera of 5 micecollected 14 days post secondary (Table 2). The nonimmune controls werenot vaccinated. The initial dilutions used on sera from test groups was1:1600.

The results were similar to those in the previous Example. Again RC-553had titers significantly higher than control sera receiving antigenalone (Table 2). The increase in titers was also reflected in theenhanced IgG2a and IgG2b responses. These titers were equivalent tothose expressed by control groups receiving MPL® immunostimulant.

TABLE 2 Comparison of Low Pyrogen Adjuvants with an Influenza VaccinePyrogen- Serum Titer Groups icity^(a) IgG IgG1 IgG2a IgG2b Nonimmune —<100 <100 <100 <100 TEoA Vehicle N.T. 12,800 51,200 1600 <1600MPL ®-TEoA 2–3 102,400 102,400 25,600 12,800 RC-553-TEoA 0.3 51,200102,400 25,600 6400 ^(a)The pyrogenicity data represents the total risein ° C. of 3 rabbits following i.v. administration of a 10 μg/Kg dose.In the pyrogen assay the compounds were solubilized in 10% EtOH/WFI(USPWater for Injection) at 100 μg/ml and then diluted with 5% dextrose inwater. N.T. means the compound was not tested.

Example 4 Adjuvant Activity Towards HBsAg

Groups of BALB/c mice injected subcutaneous with 2.0 μg HBsAg(RheinAmericana, & Rhein Biotech) ±25 μg adjuvant (MPL® immunostimulant orRC-553) on day 0 and day 21. IgG1 and IgG2a isotype titers to HBsAg weredetermined by ELISA from pooled sera collected 21 days post secondary(Table 3). The nonimmune controls were not vaccinated. In thisexperiment, RC-553 mediated increased titers compared to the controlgroup, which received antigen in PBS. RC-553 stimulated titersequivalent to the positive controls, MPL® immunostimulant (Table 3).

TABLE 3 Comparison of Low Pyrogen Adjuvants with HBsAg Serum TitersGroups Pyrogenicity^(a) IgG1 IgG2a Nonimmune — <100 <100 PBS ControlN.T. 64,000 4000 MPL ®-TEoA 2–3 128,000 1,024,000 RC-553-TEoA 0.3 32,0002,048,000 ^(a)The pyrogenicity data represents the total rise in ° C. of3 rabbits following i.v. administration of a 10 μg/Kg dose. In thepyrogen assay the compounds were solubilized in 10% EtOH/WFI(USP Waterfor Injection) at 100 μg/ml and then diluted with 5% dextrose in water.N.T. means the compound was not tested.

Example 5 CTL Activity is Increased with RC-553 Towards HBsAg ImmunizedMice

Some mice from each group of Example 4 were also used as spleen celldonors in order to evaluate CTL activity. HBsAg directed specific lysiswas assessed in a standard four hour ⁵¹Cr-release assay (Moore et al.,(1988) Cell 55: 777–785). Single cell suspensions were prepared from thespleens of mice 9 days post-vaccination. The spleen cells were treatedwith tris-buffered NH₄Cl to remove erythrocytes and resuspended at aconcentration of 7.5×10⁶/ml in RPMI/10% FCS supplemented with 5 mMHEPES, 4 mM L-glutamine, 0.05 mM 2-mercaptoethanol and antibiotics. Asynthetic peptide representing a known MHC class I, L^(d)-restricted CTLepitope (IPQSLDSWWTSL) was added to the cells at a final concentrationof 75 nM. After a four day incubation, the cells were recovered andassessed for CTL activity. Specific killing was measured against⁵¹Cr-labeled transfected P815S cells expressing the L^(d)-restrictedepitope. The target cells were a transfected P815 cell line (P815S)which express the L^(d)-restricted CTL epitope. Non-specific lysis was<10% at an E:T of 50:1 against P815 target (Table 4) In contrast to theantibody response, RC-553 stimulated significantly elevated levels ofCTL activity compared to the antigen only controls (Table 4).

TABLE 4 Comparison of Low Pyrogen Adjuvants with HBsAg Percent SpecificKilling Pyrogen- (Effector:Target Ratio) Groups icity^(a) 50:1 25:112.5:1 6.25:1 Nonimmune — 6 3 1 0 PBS N.T. 29 20 11 7 MPL ®-TEoA 2–3 8071 47 32 RC-553-TEoA 0.3 85 77 53 37 ^(a)The pyrogenicity datarepresents the total rise in ° C. of 3 rabbits following i.v.administration of a 10 μg/Kg dose. In the pyrogen assay the compoundswere solubilized in 10% EtOH/WFI(USP Water for Injection) at 100 μg/mland then diluted with 5% dextrose in water. N.T. means the compound wasnot tested.

Example 6 Ex Vivo Cytokine Induction by RC-553

The effects of RC-553 on the elaboration of TNF-α and IL-1β was measuredunder ex vivo conditions on human peripheral blood mononuclear cells.MPL® immunostimulant and RC-553 were formulated in aqueous solutions of0.2% TEoA/WFI.

Human whole blood was used to evaluate the ability of glycolipids (AGPs)to induce proinflammatory cytokines. Human whole blood is collected intoheparinized tubes and 0.45 ml of whole blood is admixed with 0.05 mlphosphate buffered saline (PBS, pH 7.4) containing the glycolipid (i.e.,the test compounds). The tubes are incubated for 4 hr at 37° C. on ashaker apparatus. The samples are then diluted with 1.5 ml sterile PBSand centrifuged. The supernatants are removed and analyzed for cellassociated TNF-α and IL-1β by sandwich ELISA using R&D Systems'Quantikine immunoassay kits for human TNF-α and IL-1β.

At 1, 5 and 10 μg/ml in the assay, RC-553 did not produce levels ofTNF-α that could be detected under the condition of the assay. Incontrast, the positive control LPS was an effective stimulator of TNF-αsecretion from the cells at 1 ng/mL. MPL® immunostimulant was effectiveat inducing TNF-α in the concentration range of 100 to 10,000 ng/mL.

Similarly, RC-553 (at 1, 5, and 10 μg/ml) did not produce detectablelevels of IL-1β. To compare the effects of RC-553, the level of IL-1βinduced with MPL® immunostimulant was assigned a value of 1 and relativeinduction of cytokines for RC-553 was ≦0.05.

Discussion of Examples 2–6

The data from these studies indicate that RC-553 is able to enhanceimmunity to vaccine antigens. RC-553 enhanced serum titers to twodistinct vaccine antigens, influenza and hepatitis surface antigens.Like MPL® immunostimulant, the RC-553 mediated a shift in the antibodyprofile from a response dominated by the IgG1 isotype to a response withhigh levels of IgG2a antibodies. In addition to enhancing the antibodyresponse, RC-553 is a good adjuvant for inducing CTL activity.

A remarkable feature of the results in this study is that RC-553 appearsto be influencing the response without inducing detectable levels of theinflammatory cytokines TNF-α or IL-1β. These cytokines are both producedby cells of the innate immune system in response to bacterial cell wallproducts including lipid A. Since RC-553 shares structural similaritieswith lipid A it is conceivable that it would also stimulate TNF-α orIL-1β and indeed many of AGP molecules do. As inflammatory cytokinesTNF-α and IL-1β stimulate the release of cascades of other cytokinemediators responsible for activating phagocytic cells and mobilizingspecific immunity. IL-1 was initially called endogenous pyrogen becauseit induces a fever response. Thus, the lack of detectable IL-1 followingadministration of RC-553 coincides with the apparent lack of fever inthe rabbit pyrogen test.

It remains possible that RC-553 in these studies actually promotes thesecretion of TNF-α and IL-1β at levels high enough to mediate activationof specific immunity yet too low to be detected in the ex vivo cytokineassay. Another option would be that these compounds stimulate cytokinemediators other than TNF-α and IL-1β that lead to a specific immuneresponse to co-administered vaccine antigens. It seems likely that IFNγis being produced. This cytokine is thought to be responsible forinducing the isotype switch to antibodies of the IgG2a subclass as wellas being a promoter of TH-1 driven CTL responses. Thus the increasedIgG2a titers and the active CTL populations both reflect the productionof IFNγ.

Example 7 Inducible Nitric Oxide Synthase (iNOS) Stimulation by RC-553

This example illustrates the effects of various glycolipids on iNOSinduction in J774 murine macrophages. The murine macrophage cell lineJ774 can be primed by IFN-γ in vitro and is very responsive tosubsequent LPS stimulation of iNOS upregulation as measured by astandard Greiss reagent ELISA assay procedure. The assay utilizes J774cells seeded at 1×10⁶/mL with 30 mL/flask and with IFN-γ added at 100units/mL for 16–24 hrs. Cells are then harvested and washed andresuspended at 2×10⁵/well in a 96-well plate and allowed to adhere.Glycolipid compounds are serially diluted into the wells for a testgroup and the resulting cultures are incubated for another 36–40 hrsbefore culture supernatants are collected from Greiss reagent analysisof nitrite release (Green et al. (1982) Anal. Biochem. 126: 131–138).Nitrite content closely parallels iNOS function.

Potency was determined as the concentration (ng/mL) of glycolipid inculture capable of inducing one-half maximal induction of nitrite(ED₅₀). The lower the ED₅₀ number, the greater the potency for iNOSinduction. The ED50 was cacluated according to methods set out inJohnson et al., (1999) J Med Chem. 42: 4640–4649.

MPL® immunostimulant was found to have an ED₅₀ of about 2 ng/mLresulting in high levels of nitrite elaboration while RC-553 exhibited anominal ED₅₀ of about ≧3000 (ng/ml).

The very low maximal iNOS activity observed with RC-553 suggests that itis essentially inactive in this system for iNOS induction.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A compound having the formula of:

and pharmaceutically acceptable salts thereof, wherein X is a memberselected from the group consisting of —O— and —NH—; Y is a memberselected from the group consisting of —O— and —S—; R¹, R² and R³ areeach members independently selected from the group consisting of(C₂–C₂₄)acyl; R⁴ is a member selected from the group consisting of —Hand —PO₃R⁷R⁸, wherein R⁷ and R⁸ are each members independently selectedfrom the group consisting of —H and (C₁–C₄)alkyl; R⁵ is a memberselected from the group consisting of —H, —CH₃ and —PO₃R⁹R¹⁰, wherein R⁹and R¹⁰ are each members independently selected from the groupconsisting of —H and (C₁–C₄)alkyl; R⁶ is selected from H, OH,(C₁–C₄)alkoxy, —PO₃R¹¹R¹², —OPO₃R¹¹R¹², —SO₃R¹¹, —OSO₃R¹¹, —NR¹¹R¹²,—SR¹¹, —CN, —NO₂, —CHO, —CO₂R¹¹, and —CONR¹¹R¹², wherein R¹¹ and R¹² areeach independently selected from H and (C₁–C₄)alkyl, with the provisothat when R⁴ is —PO₃R⁷R⁸, R⁵ is other than —PO₃R⁹R¹⁰; wherein “*¹”,“*²”, “*³” and “**” represent chiral centers; wherein the subscripts n,m, p and q are each independently an integer from 0 to 6, with theproviso that the sum of p and m is from 0 to
 6. 2. The compound of claim1, wherein X and Y are —O—, R⁴ is PO₃R⁷R⁸, R⁵ and R⁶ are H, and thesubscripts n, m, p, and q are integers from 0 to
 3. 3. The compound ofclaim 2, wherein R⁷ and R⁸ are —H.
 4. The compound of claim 3,subscripts n, m, p, and q are from 0 to
 2. 5. The compound of claim 3,wherein subscript n is 1, subscript m is 2, and subscripts p and q are0.
 6. The compound of claim 5, wherein R₁, R₂, and R₃ are tetradecanoylresidues.
 7. The compound of claim 5, wherein *¹, *², and *³ are in theR configuration.
 8. The compound of claim 5, wherein Y is in theequatorial position.
 9. The compound of claim 5, wherein ** is in the Sconfiguration.
 10. The compound of claim 5, wherein *¹, *², and *³ arein the R configuration, wherein Y is in the equatorial position, andwherein ** is in the S configuration.
 11. A pharmaceutical compositioncomprising: a therapeutically effective amount of a compositioncomprising a pharmaceutically acceptable carrier and a compound havingthe formula of:

and pharmaceutically acceptable salts thereof, wherein X is a memberselected from the group consisting of —O— and —NH—; Y is a memberselected from the group consisting of —O— and —S—; R¹, R² and R³ areeach members independently selected from the group consisting of(C₂–C₂₄)acyl; R⁴ is a member selected from the group consisting of —Hand —PO₃R⁷R⁸, wherein R⁷ and R⁸ are each members independently selectedfrom the group consisting of —H and (C₁–C₄)alkyl; R⁵ is a memberselected from the group consisting of —H, —CH₃ and —PO₃R⁹R¹⁰, wherein R⁹and R¹⁰ are each members independently selected from the groupconsisting of —H and (C₁–C₄)alkyl; R⁶ is selected from H, OH,(C₁–C₄)alkoxy, —PO₃R¹¹R¹², —OPO₃R¹¹R¹², —SO₃R¹¹, —OSO₃R¹¹, —NR¹¹R¹²,—SR¹¹, —CN, —NO₂, —CHO, —CO₂R¹¹, and —CONR¹¹ ¹², wherein R¹¹ and R¹² areeach independently selected from H and (C₁–C₄)alkyl, with the provisothat when R⁴ is —PO₃R⁷R⁸, R⁵ is other than —PO₃R⁹R¹⁰; wherein “*¹”,“*²”, “*³” and “**” represent chiral centers; wherein the subscripts n,m, p and q are each independently an integer from 0 to 6, with theproviso that the sum of p and m is from 0 to
 6. 12. The pharmaceuticalcomposition of claim 11, wherein X and Y are —O—, R⁴ is PO₃R⁷R⁸, R⁵ andR⁶ are H, and the subscripts n, m, p, and q are integers from 0 to 3.13. The pharmaceutical composition of claim 12, wherein R⁷ and R⁸ are—H.
 14. The pharmaceutical composition of claim 13, subscripts n, m, p,and q are from 0 to
 2. 15. The pharmaceutical composition of claim 13,wherein subscript n is 1, subscript m is 2, and subscripts p and q are0.
 16. The pharmaceutical composition of claim 15, wherein R₁, R₂, andR₃ are tetradecanoyl residues.
 17. The compound of claim 15, wherein *¹,*², and *³ are in the R configuration.
 18. The compound of claim 15,wherein Y is in the equatorial position.
 19. The compound of claim 15,wherein ** is in the S configuration.
 20. The compound of claim 15,wherein *¹, *², and *³ are in the R configuration, wherein Y is in theequatorial position, and wherein ** is in the S configuration.
 21. Thepharmaceutical composition of claim 11, wherein the pharmaceuticalcomposition further comprises at least one antigen.
 22. Thepharmaceutical composition of claim 21, wherein the antigen is derivedfrom the group consisting of Herpes Simplex Virus type 1, Herpes Simplexvirus type 2, Human cytomegalovirus, HIV, Hepatitis A, B, C or E,Respiratory Syncytial virus, human papilloma virus, Influenza virus,Tuberculosis, Leishmaniasis, T. Cruzi, Ehrlichia, Candida, Salmonella,Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium and Toxoplasma.23. The pharmaceutical composition of claim 21, wherein the antigen is ahuman tumor antigen.
 24. The pharmaceutical composition of claim 23,wherein the tumor antigen is derived from a prostate, colon, breast,ovarian, pancreatic, brain, head and neck, melanoma, leukemia orlymphoma cancer.
 25. The pharmaceutical composition of claim 21, whereinthe antigen is a self antigen.
 26. The pharmaceutical composition ofclaim 25, wherein the self antigen is an antigen associated with anautoimmune disease.
 27. The pharmaceutical composition of claim 26,wherein the autoimmune disease is type 1 diabetes, multiple sclerosis,myasthenia gravis, rheumatoid arthritis or psoriasis.
 28. Thepharmaceutical composition of claim 11, in an aqueous formulation. 29.The pharmaceutical composition of claim 28, wherein the aqueousformulation comprises one or more surfactants.
 30. The pharmaceuticalcomposition of claim 28, wherein the aqueous formulation comprises oneor more phospholipid surfactant.
 31. The pharmaceutical composition ofclaim 30, wherein the surfactant is selected from the group consistingof diacyl phosphatidyl glycerols, diacyl phosphatidyl cholines, diacylphosphatidic acids, and diacyl phosphatidyl ethanolamines.
 32. Thepharmaceutical composition of claim 30, wherein the surfactant isselected from the group consisting of dimyristoyl phosphatidyl glycerol(DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), distearoylphosphatidyl glycerol (DSPG), dimyristoyl phosphatidylcholine (DPMC),dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine(DSPC); dimyristoyl phosphatidic acid (DPMA), dipalmitoyl phosphatidicacid (DPPA), distearoyl phosphatidic acid (DSPA); dimyristoylphosphatidyl ethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine(DPPE) and distearoyl phosphatidyl ethanolamine (DSPE).
 33. Thepharmaceutical composition of claim 11, in an emulsion formulation.